Advances in biotechnology have made it possible to produce large quantities of macromolecules such as nucleic acids, proteins and peptides in a variety of host cells. But disadvantageously, the extraction and isolation of these macromolecules from their host cells has thus far been a multi step process, involving a first step, lysis, to free the macromolecules from their cellular confines, and then one or more subsequent steps to separate the target product from other cellular components.
A variety of techniques have been used to lyse cells, each having certain advantages and disadvantages. One such technique is mechanical or physical disruption of cell membranes. For example, sonication, French cell press, homogenization, grinding, freeze-thaw lysis, and various other methods of physically or mechanically lysing cells have been employed. But mechanical lysis requires specialized equipment that may not be readily available and is also extremely labor intensive. Equally, sonication generates heat that may denature the target polypeptide or protein. Each of these mechanical or physical techniques also results in a relatively low yield of the target product. Moreover, mechanical or physical lysis steps are also difficult to automate and miniaturize for the purpose of purifying small amounts of several proteins in parallel.
Enzymes and detergents have also been used to enzymatically or chemically lyse cells. But as with mechanical and physical lysis, enzymatic or chemical lysis also has several inherent drawbacks. Often the addition of an enzyme or detergent solution results in a dilution of the solution containing the cells to be lysed. In addition, the desired product must still be separated from the resulting membrane fragments, undesired proteins, and other cellular debris. For example, two widely utilized kits for detergent-aided purification include BugBuster® (Novagen) and B-PER (Pierce). Both of these kits use a detergent solution to disrupt the cell membrane and resultantly, release the cellular components including the target product. But neither method couples a purification step with the lysis step. The BugBuster® product, in fact, utilizes a benzonase nuclease to decrease the viscosity in the lysate due to the large amounts of chromosomal DNA present in the sample after lysis. But the product does not include any method for removal of the small DNA fragments that are necessarily generated by the nuclease digestion. The B-PER product is solely intended as an extraction system. The system includes a centrifugation step, which removes some insoluble debris; however, there is no subsequent purification of the target product from the rest of the cellular material. Any contamination of the lysates generated with the B-PER product must be removed using separate methods of purification. Analogous to mechanical or physical lysis, enzymatic or chemical lysis, as detailed above, is often labor intensive, and may result in relatively low target product yields.
Utilizing current technology, after the target product has been released from the cell by lysis, as detailed above, it is then typically purified from other cellular components. A variety of affinity capture methods have been utilized to purify proteins, peptides and nucleic acids. U.S. Pat. Nos. 4,569,794, 5,310,663, and 5,594,115 describe the use of metal chelating peptides, which include histidine residues, and their use in protein purification. Alternatively, U.S. Pat. Nos. 4,703,004, 4,851,341, 5,011,912, and 6,461,154 describe the antigenic FLAG® peptide, and the purification of proteins comprising the peptide. U.S. Pat. No. 5,654,176 describes the use of glutathione-S-transferase for the purification of proteins. U.S. Pat. No. 5,998,155 describes the use of an avidin/biotin capture system. In each of these instances, the interaction between an affinity tag or sequence on the target product and the corresponding ligand results in the “capture” of the target product. Unbound compositions and other cellular debris can then be washed away, leaving the target product bound to the tag or sequence-specific ligand. A specific eluant is then used to release the bound target product, resulting in a purified target product.
But disadvantageously, the multiple steps involved in first lysing a host cell and then purifying the target product increases the cost and time required for isolating the product, especially in high throughput applications. Moreover, in addition to being labor intensive, current lysis techniques often result in a relatively low yield of target product.